The protein kinases that constitute mitogen activated protein kinase (MAPK) pathways are of interest for their central role in mediating cellular responses to stimuli such as growth factors and cytokines. The MAPK kinase, MEK1, is activated by a phosphorylation signaling cascade in response to hormones and growth factors. Known oncogenes such as Ras and Raf are upstream activators of MEK1 and MEK1 is aberrantly activated in multiple common tumor types. Inhibition of MEK1 reverses cellular transformation. Hence, there is considerable interest in the role MEK1 signaling plays in oncogenic transformation and in targeting MEK1 for cancer therapies using, for example, small molecules. Structure assisted drug design is a tool used to optimize the success of identifying such therapeutic compounds. However, use of this powerful methodology requires three-dimensional structural information (e.g., as obtained via X-ray diffraction of the target protein). The crystal structures of unphosphorylated MEK1 have been reported, by Chen et al., in a ternary complex with ATP and 5-bromo-N-(2,3-dihydroxyl-propoxy)-3,4-difluoro-2(2-fluoro-4-iodo-phenylamino)-benzamide or {5-[3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-1,3,4-oxadiazol-2-yl}-(2-morpholin-4-yl-ethyl)-amine (“Chen crystal”; see EP1321518A1; WO 2003/54180 or U.S. 2003/0224500). This crystals suffers from several drawbacks. For example, the ATP and the benzamide compound in the MEK1 crystal disclosed by Chen at al.
(Cpd.3)) is extremely difficult to displace, e.g., by soaking, with another compound. The Chen crystal is likely not soakabie because cpd.3 is an allosteric compound that makes a complex network of interactions with both the MEK1 protein and any nucleotide bound to MEK1. The inhibitor in the Chen crystal binds to MEK1, along with a nucleotide, with particularly high affinity making its replacement with any other compound, by soaking, impractical. This shortcoming limits the utility of this Chen crystal in identifying new therapeutically useful MEK1 inhibitors. Moreover, the crystal disclosed by Chen of al. comprises a large N-terminal deletion. The deleted region comprises an α-helical region important to the negative regulation of MEK1. The crystal structures revealed in the crystals of the present invention indicate that the region deleted in the Chen crystal is an integral part of MEK1. The deleted negative-regulatory domain makes the biological accuracy and relevance of any structural data obtained from the crystal less certain.
There remains a need in the art for crystals of MEK1 into which various inhibitors can be soaked for the purpose of evaluating the inhibitor as well as obtaining structural data for the purpose of performing structure-based drug design.